Small Molecule Inhibitors of PFKFB3 and Glycolytic Flux and Their Methods of Use as Anti-Cancer Therapeutics

ABSTRACT

Small molecule inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) having the following formula: Formula (1) are provided herein. Also provided herein are pharmaceutical compositions containing Formula I compounds, together with methods of treating cancer, methods of inhibiting PFK.FB3 enzymatic activity, methods of inhibiting glycolytic flux, and methods of treating tumors by administering an effective amount of a Formula I compound.

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/306,759, filed Feb. 22, 2010, whichapplication is hereby incorporated by reference in its entirety.

The presently-disclosed subject matter relates to small-moleculeinhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) and methods of using the same. In particular, thepresently-disclosed subject matter relates to substituted benzindoles asPFKFB3 inhibitors and methods of using these inhibitors to reducecellular glycolytic flux and/or treat cancer and tumors in mammals.

The glycolytic pathway is a ten-step series of reactions that forms themajor metabolic pathway in nearly all organisms. Flux through theglycolytic pathway is adjusted in response to conditions both inside andoutside the cell. Irreversible glycolytic reactions are those catalyzedby hexokinase, phosphofructokinase, and pyruvate kinase. In metabolicpathways, such enzymes are potential targets for control, and all threeenzymes serve this purpose in glycolysis. The PFKFB enzymes (PFKFB 1-4)synthesize fructose-2,6-bisphosphate (F2,6BP) which activates6-phosphofructo-1-kinase (PFK-1), an essential control point in theglycolytic pathway.

Neoplastic cells preferentially utilize glycolysis to satisfy theirincreased needs for energy and biosynthetic precursors. Malignant tumorcells have glycolytic rates that are up to 200 times higher than thoseof their normal tissues of origin. One cancer attack strategy has beento treat cancer by starving cancerous cells in various ways. Despitegreater understanding and pharmaceutical advances in the diagnosis andtreatment of cancer, it is still estimated that nearly 13% of all humandeaths last year were due to cancer. Thus, there remains a need foradditional anti-cancer therapeutics, particularly those which targetneoplastic cells via mechanisms over-expressed in cancer cells, such asglycolytic flux, which is increased in cancer cells.

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, or PFKFB3, is avaluable molecular target for the development of anti-cancertherapeutics. Accordingly, provided herein are novel small moleculeinhibitors of PFKFB3 having the following formula:

In another embodiment, a pharmaceutical composition for the treatment ofcancer is provided, the composition comprising a Formula I compound andat least one pharmaceutically acceptable carrier.

In another embodiment, a method of treating cancer is provided, themethod comprising administering to a subject in need thereof aneffective amount of a Formula I compound.

In another embodiment, a method of treating a tumor is provided, themethod comprising administering to a subject in need thereof aneffective amount of a Formula I compound.

In still another embodiment, a method of inhibiting glycolytic flux in acell is provided, the method comprising contacting the cell with aneffective amount of a Formula I compound.

In another embodiment, a method of inhibiting enzymatic activity ofPFKFB3 in a cell is provided, the method comprising contacting the cellwith an effective amount of a Formula I compound.

These and other objects, features, embodiments, and advantages willbecome apparent to those of ordinary skill in the art from a reading ofthe following detailed description and the appended claims. Allpercentages, ratios and proportions herein are by weight, unlessotherwise specified. All temperatures are in degrees Celsius (° C.)unless otherwise specified.

FIG. 1 shows the time vs. plasma concentration pharmacokinetic profileof ACT-PFK-065 in BalbC mice (IV dosing, 10 mg/kg; IP dosing, 10 mg/kg;PO dosing, 20 mg/kg).

FIG. 2 shows the time vs. plasma concentration pharmacokinetic profileof ACT-PFK-095 in male Sprague-Dawley rats (IV dosing, 5 mg/kg; IPdosing, 10 mg/kg; PO dosing, 10 mg/kg).

FIG. 3 shows the time vs. plasma concentration pharmacokinetic profileof ACT-PFK-112 in male beagle dogs (IV dosing: 6 mg/kg; PO dosing, 24mg/kg).

FIG. 4 shows the average tumor volume as a function of time for controlgroup and treatment groups in the Lewis Lung Carcinoma (ACT-PFK-112dosed IV at 50 mg/kg using 2 different schedules that are listed on thegraph and dosed PO daily at 150 mg/kg).

FIG. 5 shows the average tumor volume as a function of time for controlgroup and treatment group in the CT-26 colon carcinoma model(ACT-PFK-112 dosed IV at 40 mg/kg using the schedule specified on thegraph).

FIG. 6 shows the average tumor volume as a function of time for controlgroup and treatment group in the HCT-116 tumor model (ACT-PFK-095 dosedIP at 40 mg/kg and PO at 80 mg/kg).

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers and optical and stereoisomers, aswell as racemic mixtures where such isomers and mixtures exist.

The term “cancer” as used herein refers to diseases caused byuncontrolled cell division and the ability of cells to metastasize, orto establish new growth in additional sites. The terms “malignant,”“malignancy,” “neoplasm,” “tumor” and variations thereof refer tocancerous cells or groups of cancerous cells.

The term “anti-cancer agent,” “anti-cancer compound,” “anti-neoplasticcompound,” “anti-tumor agent,” “anti-cancer therapeutic” and variationsthereof as used herein refer to compounds that can prevent theproliferation of cancer cells and tumors or kill cancer cells.

Specific types of cancer include, but are not limited to, skin cancers,connective tissue cancers, adipose cancers, breast cancers, lungcancers, stomach cancers, pancreatic cancers, ovarian cancers, cervicalcancers, uterine cancers, anogenital cancers, kidney cancers, bladdercancers, colon cancers, prostate cancers, central nervous system (CNS)cancers, retinal cancer, blood, and lymphoid cancers.

The term “competitive inhibitor” refers to an inhibitor whose binding toan enzyme prevents the binding of the enzyme's normal substrate.

As used herein, the term “alkyl” refers to C₁₋₂₀ inclusive, linear(i.e., “straight-chain”), branched, or cyclic, saturated or at leastpartially and in some cases fully unsaturated (i.e., alkenyl andalkynyl)hydrocarbon chains, including for example, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl,ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl,propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to (C₁-C₆) linearor branched alkyls. In other specific embodiments, “alky” comprises(C₁-C₃) linear or branched alkyl.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halogen, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl. In certain embodiments, “cycloalkyl” comprises (C₃-C₈)cycloalkyl.

The term “aryl” is used herein to refer to an aromatic substituent thatcan be a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene moiety. The common linking groupalso can be a carbonyl, as in benzophenone, or oxygen, as indiphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered hydrocarbon and heterocyclic aromatic rings. In specificembodiments, aryl comprises (C₆-C₁₄) aryl ring structures.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR′R″, wherein R′ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “substituted aryl” includes aryl groups,as defined herein, in which one or more atoms or functional groups ofthe aryl group are replaced with another atom or functional group,including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroaryl” is used herein to refer to aromatic ringstructures comprising at least one heteroatom as a ring member.Heteroaryl rings are monocyclic or fused polycyclic ring systems.Monocyclic heteroaryl rings contain from 5 to 9 member atoms (carbon andheteroatoms), more specifically 5 or 6 member atoms in the ring.Polycyclic heteroaryl rings contain from 8 to 13 member atoms, morespecifically 8 to 12 member atom in the ring. Polycyclic heteroarylrings include ring systems wherein at least one ring is heteroaryl (thesecond ring may be aryl, heteroaryl, or cycloalkyl). In specificembodiments, bicyclic heteroaryl ring systems comprise 5-, 6-, or7-membered rings fused to 5-, 6-, or 7-membered rings. In other specificembodiments, heteroaryl comprises (C₁-C₁₃) heteroaryl.

Heteroaryl rings may be substituted with at least one substituent on thering. In certain embodiments, heteroaryl rings may be substituted withhalogen, cyano, nitro, hydroxy, amino, alkyl, lower alkenyl, loweralkynyl, heteroalkyl, aryloxy, alkoxy, methylenedioxy, thioalkoxy,thioaryloxy, or any combination thereof.

Specific examples of aryl groups, including heteroaryl groups, include,but are not limited to, cyclopentadienyl, phenyl, furan, thiophene,pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole,isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline,isoquinoline, indole, carbazole, oxazole benzimidazole, azaindole, andthe like.

“Heteroatom” refers to a non-carbon atom as a ring member in aheteroaryl ring structure. In certain embodiments, the heteroatom can benitrogen, oxygen, or sulfur. Heteroatoms can contain furthersubstituents, such that in certain embodiments, heteroaryl rings cancomprise ring members selected from the group consisting of N, N═O, NH,N—(C₁-C₆)-alkyl, O, or S. In certain embodiments, a heteroaryl cancontain from 1 to 4 heteroatoms in the heteroaryl ring. In otherembodiments, a heteroaryl can contain 1, 2, 3, or 4 heteroatoms in theheteroaryl ring. In a very specific embodiment, a heteroaryl can contain1 heteroatom in the heteroaryl ring.

In specific embodiments, heteroaryl ring structures are aza ringstructures. As used herein, the term “aza” refers to a heterocyclic ringstructure containing at least one nitrogen atom. Specific examples ofaza groups include, but are not limited to, pyrrolidine, piperidine,quinuclidine, pyridine, pyrrole, indole, purine, pyridazine, pyrimidine,and pyrazine.

The term “azaaryl” refers to a heterocyclic aryl group wherein one ormore of the atoms of the aryl group ring or rings is nitrogen. Examplesof azaaryl groups include monocyclic or bicyclic mono- or diazaaryl(i.e., an aryl group comprising two nitrogen atoms), which isunsubstituted or substituted by a member selected from the groupconsisting of lower alkyl, for example methyl, lower alkoxy, for examplemethoxy, and/or halogen, for example chlorine or bromine. Therefore, theterm “azaaryl” refers to groups including, but not limited to, pyridine,pyridazine, pyrimidine, pyrazine, quinoline, quinaldine, quinoxaline,and substituted analogs thereof. In some embodiments, the azaaryl groupis pyridyl, for example 2-, 3- or 4-pyridyl; quinolinyl orisoquinolinyl, for example 4-quinolinyl or 1-isoquinolinyl; imidazolyl;pyrimidinyl, for example 2- or 4-pyrimidinyl; pyridazinyl, for example3-pyridazinyl; or pyrazinyl, for example 2-pyrazinyl.

“Alkoxy” or “alkoxyl” refer to an alkyl-O-group wherein alkyl is aspreviously described. The term “alkoxy” as used herein can refer to, forexample, methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, andpentoxl. The term “oxyalkyl” can be used interchangeably with “alkoxy.”In certain embodiments, alkoxy comprises (C₁-C₆) alkoxy.

In other specific embodiments, alkoxy comprises (C₁-C₃) alkoxy.

The term “carboxyl” refers to the —COOH group.

The term “hydroxyl” refers to the —OH group.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

As used herein, the term “acyl” refers to an organic carboxylic acidgroup wherein the —OH of the carboxyl group has been replaced withanother substituent (i.e., as represented by RCO—, wherein R is analkyl, aralkyl or aryl group as defined herein, including substitutedalkyl, aralkyl, and aryl groups). As such, the term “acyl” specificallyincludes arylacyl groups, such as an acetylfuran and a phenacyl group.Specific examples of acyl groups include acetyl and benzoyl.

“Aryloxyl” refers to an aryl-O-group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O-group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl.

“Dialkylamino” refers to an —NRR′ group wherein each of R and R′ isindependently an alkyl group and/or a substituted alkyl group aspreviously described. Exemplary alkylamino groups includeethylmethylamino, dimethylamino, and diethylamino.

“Alkoxycarbonyl” refers to an alkyl-O—CO-group. Exemplary alkoxycarbonylgroups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, andt-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—CO-group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO-group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an H₂N—CO-group.

“Alkylcarbamoyl” refers to a R′RN—CO-group wherein one of R and R′ ishydrogen and the other of R and R′ is alkyl and/or substituted alkyl aspreviously described.

“Dialkylcarbamoyl” refers to a R′RN—CO-group wherein each of R and R′ isindependently alkyl and/or substituted alkyl as previously described.

“Acyloxyl” refers to an acyl-O-group wherein acyl is as previouslydescribed.

“Acylamino” refers to an acyl-NR-group wherein acyl is as previouslydescribed and R is H or alkyl. Thus, the “acylamino” group can have thestructure —NR—C(═O)—R′, wherein R′ is alkyl, aryl, aralkyl, and thelike.

The term “amino” refers to the —NH₂ group.

The term “carbonyl” refers to the —(C═O)-group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” refers to a compound described previously herein whereina carbon atom is replaced by an oxygen atom.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₂-R₇, or groups A, B, D, E,G, or J), can be identical or different. For example, both R₂ and R₃ canbe the same substituent, or R₂ and R₃ can each be different substituentsselected from a specified group.

I. Compounds

In one embodiment, the PFKFB3 inhibitors of the present invention havethe following structural formula:

wherein:

A, B, D, E, G and J are independently selected from the group consistingof N or C substituted with one of R₂, R₃, R₄, R₅, R₆ or R₇, wherein ifA, B, D, E, G, or J are N, then R₂, R₃, R₄, R₅, R₆ or R₇ represent afree electron pair at the N atom;

R₁ is selected from the group consisting of hydrogen and linear orbranched (C₁-C₆)-alkyl;

R₂, R₃, R₄, R₅, R₆ and R₇ are independently of one another, whenattached to N, a free electron pair, or, when attached to C, areselected from the group consisting of hydrogen, halogen, —COOH, linearor branched (C₁-C₆)-alkyl, linear or branched (C₁-C₆)-alkoxy, hydroxyl,—NH₂, N—(C₁-C₆)-alkyl, N-di-(C₁-C₆)-alkyl, and —SO₂CH₃;

R₈ is selected from the group consisting of hydrogen and linear orbranched (C₁-C₆)-alkyl;

and

Y is selected from the group consisting of substituted or unsubstituted(C₆-C₁₄)-aryl, substituted or unsubstituted (C₁-C₁₃)-heteroaryl havingat least one of N, N═O, NH, N—(C₁-C₆)-alkyl, O, or S as ring members,and substituted or unsubstituted (C₃-C₈)-cycloalkyl.

In another embodiment of the invention, PFKFB3 inhibitor compounds havethe following structural formula, wherein A, B, D, E, G, J, X, Y, andR₁-R₈ are defined as above for Formula I:

In another embodiment of the invention, PFKFB3 inhibitor compounds havethe following structural formula, wherein A, B, D, E, G, J, X, Y, andR₁-R₈ are defined as above for Formula I:

Examples of Formula I and Formula II compounds are shown in Table 1,below.

TABLE 1 Exemplary Compounds Compound Number Mw Structure, Name and NMRData ACT-PFK-061 271.1

(1H-Benzo[g]indol-2-yl)-phenyl-methanone. The compound may be preparedusing Scheme 5. ¹H NMR (300 MHz, DMSO-d₆): δ 12.92 (s, 1 H), 8.87-8.85(m, 1 H), 7.97-7.95 (m, 3 H), 7.74-7.52 (m, 7 H), 7.26 (m, 1 H).ACT-PFK-065 271.1

(3H-Benzo[e]indol-2-yl)-phenyl-methanone. The compound may be preparedusing Scheme 2. ¹H NMR (300 MHz, DMSO-d₆) δ 12.43 (s, 1 H), 8.39 (d, J =6.0. Hz, 1 H), 8.01-7.93 (m, 3H), 7.80-7.72 (m, 2 H), 7.69-7.65 (m. 1H),7.63-7.45 (m, 4 H), 7.48-7.43 (m, 1H). ACT-PFK-093 301.3

(3H-Benzo[e]indol-2-yl)-(4-methoxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.36 (s, 1 H),8.39 (d, J = 8.1. Hz, 1 H), 8.03 (d, J = 8.7 Hz, 2H), 7.94 (d, J = 6.0Hz, 1 H), 7.81-7.75 (m. 2H), 7.64-7.56 (m, 2 H), 7.45 (s, 1 H), 7.16 (d,J = 8.4. Hz, 2H), 3.90 (s, 3H). ACT-PFK-095 272.3

(3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone. The compound may beprepared using Scheme 3. ¹H NMR (300 MHz, DMSO-d₆): δ 12.50 (s, 1 H),8.87-8.85 (m, 2 H), 8.42-8.39 (m, 1 H), 7.97-7.94 (m, 1 H), 7.88-7.86(m, 3 H), 7.83-7.80 (m, 1 H), 7.65-7.58 (m, 2 H), 7.50- 7.44 (m, 1 H)ACT-PFK- 095•HCl 308.76

HCl salt of (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone. The compoundmay be prepared using Scheme 3. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66 (s, 1H), 9.02-9.00 (m, 2 H), 8.36 (d, J = 8.1 Hz, 1 H), 8.15-8.14 (m, 2 H),7.96 (d, J = 7.8 Hz, 1 H), 7.89-7.82 (m, 2 H), 7.65-7.57 (m, 2 H),7.50-7.45 (m, 1 H). ACT-PFK-096 301.34

(3H-Benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.43 (s, 1 H),8.40 (d, J = 9.0 Hz, 1 H), 7.94 (d, J = 9.0 Hz, 1 H), 7.82-7.76 (m, 2H), 7.64-7.43 (m, 6 H), 7.27 (d, J = 6.0 Hz, 1 H), 3.88 (s, 3 H).ACT-PFK-097 272.3

(3H-Benzo[e]indol-2-yl)-pyridin-3-yl-methanone. The compound may beprepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.54 (s, 1 H),9.14 (d, J = 1.5 Hz, 1 H), 8.89-8.87 (dd, J = 4.8, 1.2 Hz, 1 H), 8.44-8.35 (m, 2 H), 7.96-7.90 (m, 2 H), 7.82-7.79 (m, 1 H), 7.69-7.56 (m, 3H), 7.47 (t, J = 7.2 Hz 1 H). ACT-PFK-098 301.34

(3H-Benzo[e]indol-2-yl)-(2-methoxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.36 (s, 1 H),8.27 (d, J = 7.8 Hz, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.77-7.74 (m, 1H), 7.62-7.41 (m, 6 H), 7.23 (d, J = 8.4 Hz, 1 H), 7.11 (t, J = 7.2 Hz,1 H), 3.77 (s, 3 H). ACT-PFK-099 287.31

(3H-Benzo[e]indol-2-yl)-(2-hydroxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 11.01 (s, 1 H),8.10 (d, J = 9.0 Hz, 1 H), 8.01-7.99 (m, 2 H), 7.63-7.46 (m, 6 H), 7.02(d, J = 9.0 Hz, 1 H), 6.91-6.86 (m, 1 H). ACT-PFK-100 287.3

(3H-Benzo[e]indol-2-yl)-(4-hydroxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.32 (s, 1 H),10.36 (s, 1 H), 8.40 (d, J = 9.0 Hz, 1 H), 7.97-7.93 (m, 3 H), 7.81-7.74(m. 2H), 7.64-7.55 (m, 2 H), 7.47-7.45 (m, 1 H), 6.99-6.96 (m, 2 H)ACT-PFK-101 285.34

(5-Methyl-3H-benzo[e]indol-2-yl)-phenyl-methanone. The compound may beprepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.32 (s, 1 H),8.40 (d, J = 7.2 Hz, 1 H), 8.04-7.97 (m, 3 H), 7.76-7.68 (m, 2 H),7.64-7.53 (m, 5 H), 2.70 (s, 3 H). ACT-PFK-102 272.30

Phenyl-(7H-pyrrolo[2,3-h]quinolin-8-yl)-methanone. The compound may beprepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.66 (s, 1 H),8.87-8.86 (m, 1 H), 8.42-8.38 (dd, J = 8.1, 1.5 Hz, 1 H), 8.00 (d, J =6.9 Hz, 2 H), 7.84-7.82 (m, 1 H), 7.75-7.64 (m, 5 H), 7.53- 7.49 (m, 1H). ACT-PFK-103 287.3

(3H-Benzo[e]indol-2-yl)-(3-hydroxy-phenyl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.40 (s, 1 H),9.88 (s, 1H), 8.38 (d, J = 8.1 Hz, 1 H), 7.94 (d, J = 8.1 Hz, 1 H),7.78-7.76 (m, 2H), 7.64-7.54 (m. 2H), 7.48-7.35 (m, 4H), 7.10-7.07 (m,1H). ACT-PFK-104 289.30

(5-Fluoro-3H-benzo[e]indol-2-yl)-phenyl-methanone. The compound may beprepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.48 (s, 1 H),8.47 (d, J = 8.4 Hz, 1 H), 8.06 (d, J = 8.1 Hz, 1 H), 7.99- 7.96 (m, 2H), 7.84 (d, J = 1.8 Hz, 1 H), 7.70-7.55 (m, 5 H), 7.38 (d, J = 11.1 Hz,1 H). ACT-PFK-105 306.7

(3H-benzo[e]indol-2-yl)-(2-chloro-pyridin-4-yl)-methanone. The compoundmay be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.75 (s, 1H), 8.91 (d, J = 8.4 Hz, 1 H), 8.84 (d, J = 5.4 Hz, 2 H), 8.02 (d, J =7.8 Hz, 1 H) 7.88-7.86 (m, 1 H), 7.72 (d, J = 5.7 Hz, 2 H), 7.69-7.61(m, 2 H), 7.57-7.55 (m, 1 H). ACT-PFK-106 288.3

(3H-benzo[e]indol-2-yl)-(1-oxy-pyridin-4-yl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.51 (s, 1 H),8.40 (d, J = 9.0 Hz, 3 H), 8.01-7.94 (m, 4 H), 7.80 (d, J = 9.0 Hz, 1H), 7.64-7.58 (m, 2 H), 7.48 (d, J = 9.0 Hz, 1 H). ACT-PFK-107 275.34

Phenyl-(6,7,8,9-tetrahydro-3H-benzo[e]indol-2-yl)-methanone. Thecompound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ11.86 (s, 1 H), 7.93 (d, J = 7.2 Hz, 2 H), 7.71-7.57 (m, 3 H), 7.25-7.22(m, 1 H), 7.03-7.00 (m, 2 H), 2.89 (brs, 2 H), 2.76 (brs, 2 H), 1.80(brs, 4 H). ACT-PFK-108 317.34

(3H-Benzo[e]indol-2-yl)-(4-hydroxy-3-methoxylthenyl)-methanone. Thecompound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ12.30 (s, 1 H), 9.98 (s, 1 H), 8.41 (d, J = 9.0 Hz, 1 H), 7.95- 7.73 (m,3 H,), 7.64-7.42 (m, 5 H), 6.99 (d, J = 6.0 Hz, 1 H), 3.90 (s, 3 H).ACT-PFK-109 407.46

(3H-Benzo[e]indol-2-yl)-(4-benzyloxy-3-methoxy-phenyl)-methanone. Thecompound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ12.35 (s, 1H), 8.42-8.40 (m, 1 H), 7.95-7.25 (m, 14 H), 5.25 (s, 2 H),3.90 (s, 3 H). ACT-PFK-110 329.35

4-(3H-Benzo[e]indole-2-carbonyl)-benzoic acid methyl ester. The compoundmay be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.53 (s, 1H), 8.39 (d, J = 8.0 Hz, 1 H), 8.20-8.09 (m, 4 H), 7.95 (d, J = 8.0 Hz,1 H), 7.84 (t, J = 9.0 Hz, 2 H), 7.65-7.56 (m, 2 H), 7.46 (t, J = 6.0Hz, 1 H), 3.94 (s, 3 H). ACT-PFK-111 315.32

4-(3H-Benzo[e]indole-2-carbonyl)-benzoic acid. The compound may beprepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 13.36 (s, 1 H),12.52 (s, 1 H), 8.40 (d, J = 8.0 Hz, 1 H), 8.18-8.07 (m, 4 H), 7.95 (d,J = 8.0 Hz, 1 H), 7.82 (t, J = 9.0 Hz, 2 H), 7.65-7.55 (m, 2 H), 7.46(t, J = 6.0 Hz, 1 H). ACT-PFK-112 286.33

(4-Amino-phenyl)-(3H-benzo[e]indol-2-yl)-methanone. The compound may beprepared using Scheme 4. ¹H NMR (300 MHz, DMSO-d₆) δ 12.18 (s, 1 H),8.38 (d, J = 9.0 Hz, 1 H), 7.92 (d, J = 9.0 Hz, 1 H), 7.86-7.83 (m, 2H), 7.76-7.70 (m, 2 H), 7.59-7.56 (m, 2 H), 7.43- 7.42 (m, 1 H), 6.69(d, J = 8.7 Hz, 2 H), 6.09 (s, 2 H). ACT-PFK-113 435.5

5-(3H-Benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methyl ester.The compound may be prepared using Scheme similar to Scheme 1. ¹H NMR(300 MHz, DMSO-d₆) δ 12.44 (s, 1 H), 8.40-8.24 (m, 3 H), 7.95 (d, J =8.1 Hz, 1 H), 7.84-7.77 (m, 2H), 7.65-7.55 (m, 4H), 7.48-7.36 (m, 5H),5.39 (s, 2H), 3.87 (s, 3H). ACT-PFK-114 421.4

5-(3H-Benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acidmethanone. Thecompound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ12.98 (br s, 1 H), 12.42 (s, 1H), 8.20- 8.45 (m, 3H), 7.96-7.76 (m, 3H), 7.64-7.38 (m, 9H), 5.38 (s, 2H). ACT-PFK-115 302.3

(3H-Benzo[e]indol-2-yl)-(2-methoxy-pyridin-4-yl)-methanone. The compoundmay be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.53 (s, 1H), 8.43-8.40 (m, 2H), 7.94 (d, J = 7.8 Hz, 1H). 7.86-7.79 (m, 2 H),7.63-7.57 (m. 2H), 7.46-7.41 (m, 2 H), 7.23 (br s, 1H), 3.97 (s, 3H).ACT-PFK-116 319.33

(5-Fluoro-3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanone. Thecompound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ12.50 (s, 1 H), 8.50 (d, J = 8.4 Hz, 1 H), 8.07 (d, J = 7.8 Hz, 1 H),7.87 (brs, 1 H), 7.72-7.65 (m, 1 H), 7.59-7.54 (m, 3 H), 7.45-7.38 (m, 2H), 7.29-7.26 (m, 1 H), 3.88 (s, 3 H). ACT-PFK-118 290.29

(5-Fluoro-3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone. The compoundmay be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.64 (s, 1H), 8.86 (d, J = 4.8 Hz, 2 H), 8.49 (d, J = 8.1 Hz, 1 H), 8.08 (d, J =8.1 Hz, 1 H), 7.93-7.85 (m, 3 H), 7.71-7.68 (m, 1 H), 7.61-7.58 (m, 1H), 7.40 (d, J = 10.8 Hz, 1 H). ACT-PFK-117 425.5

(4-Benzyloxy-3-methoxy-phenyl)-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone. The compound may be prepared using Scheme 1. ¹H NMR (300 MHz,DMSO-d₆) δ 12.41 (s, 1 H), 8.50 (d, J = 7.8 Hz, 1 H), 8.07(d, J = 7.8Hz, 1H), 7.89 (br s, 1 H), 7.72-7.69 (m. 2H), 7.59-7.38 (m, 8 H), 7.26(d, J = 8.4 Hz, 1H), 5.25 (s, 2H), 3.90 (s, 3H). ACT-PFK-119 335.33

(5-Fluoro-3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy-phenyl)-methanone. The compound may be prepared using Scheme 1. ¹H NMR (300 MHz,DMSO-d₆) δ 12.37 (s, 1 H), 9.99 (s, 1 H), 8.50 (d, J = 8.1 Hz, 1 H),8.07 (d, J = 8.1 Hz, 1 H), 7.88 (d, J = 1.8 Hz, 1 H), 7.72-7.52 (m, 4H), 7.39 (d, J = 11.1 Hz, 1 H), 6.99 (d, J = 8.1 Hz, 1 H), 3.90 (s, 3H). ACT-PFK-120 301.3

(3H-Benzo[e]indol-2-yl)-(3-hydroxymethyl-phenyl)-methanone. The compoundmay be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.46 (s, 1H), 8.36 (d, J = 7.8 Hz, 1 H), 8.09-7.94 (m, 3H), 7.81-7.78 (m, 3H),7.71-7.58 (m. 3H), 7.47 (d, J = 7.5 Hz, 1 H), 5.60 (s, 2H). ACT-PFK-121295.4

Cyclohexyl-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone. The compound maybe prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.16 (s, 1 H),8.45 (d, J = 8.7 Hz, 1 H), 8.12 (s, 1H), 8.05 (d, J = 8.10 Hz, 1H), 7.71(t, J = 7.50 Hz. 1H), 7.56 (t, J = 7.50 Hz. 1H), 7.33 (d, J = 11.4 Hz,1H), 1.95-1.68 (m, 5H), 1.56-1.37 (m, 4H), 1.34-1.17 (m, 1H).ACT-PFK-122 323.3

(5-Fluoro-3H-benzo[e]indol-2-yl)-(3-fluoro-4-hydroxy-phenyl)- methanone.The compound may be prepared using Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ12.41 (s, 1 H), 10.99 (s, 1H), 8.51 (d, J = 8.1 Hz, 1 H), 8.07 (d, J =8.1 Hz, 1 H), 7.92 (s, 1H), 7.80-7.68 m, 3H), 7.59-7.55 (m, 1 H), 7.38(d, J = 11.1 Hz, 1 H), 7.19-7.13 (m, 1H). ACT-PFK-123 285.3

(3H-Benzo[e]indol-2-yl)-p-tolyl-methanone. The compound may be preparedusing Scheme 1. ¹H NMR (300 MHz, DMSO-d₆) δ 12.44 (s, 1 H), 8.39 (d, J =7.8. Hz, 1 H), 7.09-7.96 (m, 3H), 7.56-7.80 (m, 2 H), 7.57-7.64 (m. 2H),7.42-7.45 (m, 3 H), 2.46 (s, 3H). ACT-PFK-124 C₂₀H₁₇NO₂ Mol. Wt.: 303.4

(3H-Benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanol. ¹H NMR (300 MHz,DMSO-d₆) δ 11.40 (s, 1 H), 8.13 (d, J = 6.0. Hz, 1 H), 7.69 (d, J = 7.8Hz, 1 H), 7.54-7.42 (m, 3 H), 7.35-7.23 (m. 2H), 7.12-7.04 (m, 2 H),6.84-6.79 (m, 2 H), 6.13 (d, J = 4.2, 1 H), 5.91 (d, J = 3.9, 1H), 3.75(s, 3H). ACT-PFK-125 C₁₈H₁₄N₂O Mol. Wt.: 274.3

(3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanol. ¹H NMR (300 MHz, DMSO-d₆)δ 11.50 (s, 1 H), 8.55 (d, J = 4.8. Hz, 2 H), 8.14 (d, J = 8.1 Hz, 1 H),7.86 (d J = 7.8, 1 H), 7.55-7.44 (m. 5 H), 7.36-7.31 (m, 1 H), 6.84 (s,1 H), 6.39 (br s, 1 H), 5.97 (s, 1H). ACT-PFK-126 C₁₉H₁₄N₂O Mol. Wt.:286.3

3H-Benzo[e]indole-2-carboxylic acid phenylamide. ¹H NMR (300 MHz,DMSO-d₆) δ 12.22 (s, 1 H), 10.28 (s, 1 H), 8.23 (d, J = 7.8. Hz, 1 H),8.01-7.94 (m, 2 H), 7.86-7.83 (m, 2 H), 7.71-7.59 (m. 3 H), 7.47- 7.37(m, 3 H), 7.14-7.09 (m, 1 H). ACT-PFK-127 C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

3H-Benzo[e]indole-2-carboxylic acid (3-methoxy-phenyl)-amide. ¹H NMR(300 MHz, DMSO-d₆) δ 12.22 (s, 1 H), 10.24 (s, 1 H), 8.23 (d, J = 6.3.Hz, 1 H), 8.00-7.94 (m, 2 H), 7.71-7.68 (m, 1 H), 7.63-7.58 (m. 2 H),7.52 (s, 1 H), 7.45-7.43 (m, 2 H), 7.32-7.26 (m, 1 H), 6.71-6.68 (m, 1H), 3.79 (s, 3 H). ACT-PFK-128 C₂₁H₁₈N₂O Mol. Wt.: 314.4

(3H-Benzo[e]indol-2-yl)-(4-dimethylamino-phenyl)-methanone. ¹H NMR (300MHz, DMSO-d₆) δ 12.22 (s, 1 H), 8.39-8.37 (m, 1 H), 7.99- 7.92 (m, 3 H),7.79-7.72 (m, 2 H), 7.64-7.54 (m, 2 H), 7.46-7.44 (m. 1 H), 6.87-6.84(m, 2 H), 3.31 (s, 6 H). ACT-PFK-129 C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

(4-Amino-3-methoxy-phenyl)-(3H-benzo[e]indol-2-yl)-methanone. ¹H NMR(300 MHz, DMSO-d₆) δ 12.29 (s, 1 H), 8.36 (d, J = 8.1 Hz, 1 H), 7.93 (d,J = 8.1 Hz, 1 H), 7.74 (d, J = 8.7 Hz, 1 H), 7.65-7.54 (m, 4 H),7.47-7.42 (m. 1 H), 7.05 (d, J = 8.1 Hz, 1 H), 6.73-6.68 (m, 1 H), 6.35(s, 2 H), 3.88 (s, 3 H). ACT-PFK-130 C₂₀H₁₆N₂O₃ Mol. Wt.: 332.4

(4-Amino-3-methoxy-phenyl)-(5-hydroxy-3H-benzo[e]indol-2-yl)- methanone.¹H NMR (300 MHz, DMSO-d₆) δ 11.90 (s, 1 H), 10.34 (s, 1 H), 8.25 (d, J =8.1 Hz, 1 H), 8.15 (d, J = 8.1 Hz, 1 H), 7.56-7.45 (m. 3 H), 7.43-7.38(m, 1 H), 7.07-7.01 (m. 2 H), 6.71-6.66 (m, 1 H), 6.14 (s, 2 H), 3.87(s, 3 H). ACT-PFK-131 C₂₁H₁₈N₂O₃ Mol. Wt.: 346.4

(4-Amino-3-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)- methanone.¹H NMR (300 MHz, DMSO-d₆) δ 12.09 (s, 1 H), 8.30 (d, J = 8.1 Hz, 1 H),8.17 (d, J = 8.1 Hz, 1 H), 7.60-7.53 (m, 3 H), 7.46- 7.40 (m, 1 H),7.08-7.00 (m. 2 H), 6.72-6.69 (m, 1 H), 6.20 (s, 2 H), 4.02 (s, 3 H),3.87 (s, 3 H). ACT-PFK-133 C₂₀H₁₆N₂O₃S Mol. Wt.: 364.4

N-[4-(3H-Benzo[e]indole-2-carbonyl)-phenyl]-methanesulfonamide. ¹H NMR(300 MHz, DMSO-d₆) δ 12.38 (s, 1 H), 10.37 (s, 1 H), 8.41 (d, J = 8.1Hz, 1 H), 8.05-7.93 (m, 3 H), 7.85-7.75 (m, 2 H), 7.64-7.55 (m, 2 H),7.45-7.40 (m. 3 H), 3.33 (s, 3 H). ACT-PFK-134 C₁₉H₁₅N₃O Mol. Wt.: 301.3

3H-Benzo[e]indole-2-carboxylic acid (4-amino-phenyl)-amide. ¹H NMR (300MHz, DMSO-d₆) δ 12.09 (s, 1 H), 9.92 (s, 1 H), 8.19 (d, J = 8.1 Hz, 1H), 7.95-7.91 (m, 2 H), 7.68-7.57 (m, 3 H), 7.44-7.42 (m, 3 H), 6.58 (d,J = 8.1 Hz, 2 H), 4.95 (s, 2 H). ACT-PFK-135 C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

(4-Amino-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone. ¹H NMR(300 MHz, DMSO-d₆) δ 11.99 (s, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.16 (d, J= 8.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.68 (s, 1H), 7.61-7.56 (m,1H), 7.46-7.41 (m, 1H), 7.00 (s, 1 H), 6.68 (d, J = 8.4 Hz,, 2H), 6.02(s, 2H), 4.01 (s, 3H). ACT-PFK-136 C₂₀H₁₅FN₂O₂ Mol. Wt.: 334.3

(4-Amino-2-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)- methanone.¹H NMR (300 MHz, DMSO-d₆) δ 12.05 (s, 1H), 8.37 (d, J = 6.3 Hz, 1H),8.17 (d, J = 8.7 Hz, 1H), 7.76-7.57 (m, 4H), 7.47- 7.44(m, 1H),6.99-6.89 (m, 2H), 6.09 (s, 2H), 4.01 (s, 3H). ACT-PFK-137 C₂₀H₁₅FN₂O₂Mol. Wt.: 334.3

(4-Amino-3-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-138 C₂₁H₁₈N₂O3 Mol. Wt.: 346.4

(4-Amino-2-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-139 C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

(4-Amino-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone ACT-PFK-140C₂₁H₁₈N₂O₃ Mol. Wt.: 346.4

(4-Amino-3-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-141 C₂₁H₁₈N₂O₃ Mol. Wt.: 346.4

(4-Amino-2-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-142 C₂₀H₁₅FN₂O₂ Mol. Wt.: 334.3

(4-Amino-3-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-143 C₂₀H₁₅FN₂O₂ Mot. Wt.: 334.3

(4-Amino-2-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-144 C₁₉H₁₃FN₂O Mol. Wt.: 304.3

(4-Amino-3-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone ACT-PFK-145C₁₉H₁₃FN₂O Mol. Wt.: 304.3

(4-Amino-2-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone ACT-PFK-146C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

(4-Amino-phenyl)-(7-methoxy-3H-benzo[e]indol-2-yl)-methanone ACT-PFK-147C₂₀H₁₆N₂O₂ Mol. Wt.: 316.4

(4-Amino-phenyl)-(5-hydroxy-3-methyl-3H-benzo[e]indol-2-yl)- methanoneACT-PFK-148 C₁₉H₁₄FN₃O₂ Mol. Wt.: 335.3

(7-Amino-5-fluoro-9-hydroxy-3H-benzo[e]indol-2-yl)-(3-methyl-pyridin-4-yl)-methanone ACT-PFK-149 C₁₈H₁₄N₄O₂ Mol. Wt.: 318.3

(5-Amino-3H-pyrrolo[3,2-f]isoquinolin-2-yl)-(3-methoxy-pyridin-4-yl)-methanone ACT-PFK-150 C₁₉H₁₅N₃O₂ Mol. Wt.: 317.3

(4-Amino-2-methyl-phenyl)-(9-hydroxy-3H-pyrrolo[2,3-c]quinolin-2-yl)-methanone ACT-PFK-151 C₂₀H₁₆N₂O₃S Mol. Wt.: 364.4

(4-Amino-phenyl)-(7-methanesulfonyl-3H-benzo[e]indol-2-yl)- methanone

II. Chemical Synthesis

Formula I compounds of the present invention are prepared using thegeneral method described below in Scheme I, together with syntheticmethods known by one in the art of organic synthesis and variationsthereon. One skilled in the art will appreciate that aryl and naphthylmoieties may be unsubstituted or substituted with any number of suitablesubstituents. Such variations are within the purview of the ordinaryskilled artisan

Referring to step (i) of Scheme 1, 1-naphthaldehyde or substituted1-naphthaldehyde is reacted with aryl methyl ketone under basicconditions from 0° C. to 100° C. to produce corresponding chalcones.Substituted naphthaldehydes and substituted aryl methyl ketones arepurchased from commercial sources or prepared according to literatureprocedures. In step (ii), the chalcones so produced are further reactedwith an azide source in the presence of ceric ammonium nitrate at 0° C.to 100° C. and then in step (iii), thermal cyclization at 50° C. to 200°C. of the azido compound in an appropriate solvent gives the desiredsubstituted benzindoles.

In another embodiment, Formula I compounds of the present invention arealso prepared using the method described below in Scheme 2, togetherwith synthetic methods known by one in the art of organic synthesis andvariations thereon. One skilled in the art will appreciate that phenyland naphthyl moieties may be unsubstituted or substituted with anynumber of suitable substituents. Such variations are within the purviewof the ordinary skilled artisan.

The compounds of Formula (I) can also be prepared using above Scheme 2,wherein substituted or unsubstituted 1-naphthaldehyde (3) is reactedwith azidomethyl ester (2) under basic conditions at 0° C. to 100° C. toproduce compound (4). The elimination of hydroxyl group is achievedthrough the formation of methanesulphonyl derivative followed bycyclization with heating at 100° C. to 200° C. to give compound (6).Further basic hydrolysis of ester (6) results in the corresponding acid(7) which is reacted with N,N′-carbonyldiimidazole to yield amide (8).The displacement of imidazole moiety with corresponding aryl magnesiumbromide produces the desired target (9).

In a specific embodiment, compound ACT-PFK-095 and its analogs areprepared using the method described below in Scheme 3, together withsynthetic methods known by one in the art of organic synthesis andvariations thereon:

In another specific embodiment, compound ACT-PFK-112 and its analogs areprepared using the method described below in Scheme 4, together withsynthetic methods known by one in the art of organic synthesis andvariations thereon:

In another embodiment, Formula II compounds of the present invention areprepared using the method described below in Scheme 5, together withsynthetic methods known by one in the art of organic synthesis andvariations thereon. One skilled in the art will appreciate that phenyland naphthyl rings may be unsubstituted or substituted with any numberof suitable substituents. Such variations are within the purview of theordinary skilled artisan.

III. Pharmaceutical Compositions

The compounds of Formula I, Formula II, and Formula III, includingtautomeric, enantiomeric or diastereomeric forms and pharmaceuticallyacceptable salts, prodrugs, or metabolites thereof, are all referred toherein as “anti-cancer compounds.” The compounds disclosed herein can beadministered to a subject either alone, or as part of a pharmaceuticalcomposition.

Pharmaceutical compositions comprising the aforementioned anti-cancercompounds also are provided herein. These pharmaceutical compositionscomprise active compounds as described herein, in a pharmaceuticallyacceptable carrier. Pharmaceutical formulations can be prepared fororal, intravenous, parenteral, or aerosol administration as discussed ingreater detail below. Also, the compounds of the present inventionprovide such anti-cancer compounds that can be reconstituted to formpharmaceutically acceptable compositions (including compositionspharmaceutically acceptable in humans) for administration.

The term “carrier,” as used herein, includes pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.

The therapeutically effective dosage of any specific anti-cancercompound, the use of which is within the scope of embodiments describedherein, will vary somewhat from compound to compound, and subject tosubject, and will depend upon the condition of the subject and the routeof delivery. As a general proposition, a dosage from about 0.1 to about500 mg/kg will have therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid, liquid, or gel, or can be administered intramuscularly orintravenously as a solution, suspension, or emulsion. Alternatively, thecompounds or salts also can be administered by inhalation,intravenously, or intramuscularly as a liposomal suspension. Whenadministered through inhalation the active compound or salt should be inthe form of a plurality of solid particles or droplets having a particlesize from about 0.5 to about 5 microns, and preferably from about 1 toabout 2 microns.

Pharmaceutical compositions suitable for intravenous or intramuscularinjection are further embodiments provided herein. The pharmaceuticalcompositions comprise a compound of Formula I, Formula II, or FormulaIII described herein, including tautomeric, enantiomeric ordiastereomeric forms and pharmaceutically acceptable salts, prodrugs, ormetabolites thereof, in any pharmaceutically acceptable carrier. If asolution is desired, water is the carrier of choice with respect towater-soluble compounds or salts. With respect to the water-solublecompounds or salts, an organic vehicle, such as glycerol, propyleneglycol, polyethylene glycol, or mixtures thereof, can be suitable. Inthe latter instance, the organic vehicle can contain a substantialamount of water. The solution in either instance can then be sterilizedin a suitable manner known to those in the art, and typically byfiltration through a 0.22-micron filter. Subsequent to sterilization,the solution can be dispensed into appropriate receptacles, such asdepyrogenated glass vials. The dispensing is preferably done by anaseptic method. Sterilized closures can then be placed on the vials and,if desired, the vial contents can be lyophilized.

In addition to compounds of Formula I, Formula II, or Formula III,including tautomeric, enantiomeric or diastereomeric forms andpharmaceutically acceptable salts, prodrugs, or metabolites thereof, thepharmaceutical compositions can contain other additives, such aspH-adjusting additives. In particular, useful pH-adjusting agentsinclude acids, such as hydrochloric acid, bases or buffers, such assodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodiumborate, or sodium gluconate. Further, the compositions can containantimicrobial preservatives. Useful antimicrobial preservatives includemethylparaben, propylparaben, and benzyl alcohol. The antimicrobialpreservative is typically employed when the formulation is placed in avial designed for multi-dose use. The pharmaceutical compositionsdescribed herein can be lyophilized using techniques well known in theart.

In yet another embodiment of the subject matter described herein, thereis provided an injectable, stable, sterile formulation comprising acompound of Formula I, Formula II, Formula III, or a tautomeric,enantiomeric or diastereomeric form or pharmaceutically acceptable salt,prodrug, or metabolites thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate, which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid formulationsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundsalt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent, which is physiologicallyacceptable, can be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier.

Other pharmaceutical compositions can be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art.

Pharmaceutical compositions also are provided which are suitable foradministration as an aerosol by inhalation. These compositions comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired composition can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles should have a particle size in the range of about 0.5 to about10 microns, more preferably from about 0.5 to about 5 microns. The solidparticles can be obtained by processing the solid compound or a saltthereof, in any appropriate manner known in the art, such as bymicronization. Most preferably, the size of the solid particles ordroplets will be from about 1 to about 2 microns.

As indicated, both water-soluble and water-insoluble active compoundsare provided. As used herein, the term “water-soluble” is meant todefine any composition that is soluble in water in an amount of about 10mg/mL, or greater. Also, as used herein, the term “water-insoluble” ismeant to define any composition that has solubility in water of lessthan about 1 mg/mL. In some embodiments, water-soluble compounds orsalts can be desirable whereas in other embodiments water-insolublecompounds or salts likewise can be desirable.

IV. Methods of Inhibiting Cell Proliferation and Treating Cancer

The compounds of the present invention and compositions including themare useful for inhibiting cell proliferation and/or treating cancer.

In some embodiments, the methods for inhibiting cell proliferation ortreating a cancer comprise administering to a subject in need thereof ananti-cancer compound as described herein. These active compounds, as setforth above, include the compounds of Formula I, Formula II, and FormulaIII, including tautomeric, enantiomeric or diastereomeric forms andpharmaceutically acceptable salts, prodrugs, or metabolites thereof. Insome embodiments, the active compound is present in a pharmaceuticalformulation as described hereinabove.

The presently disclosed compounds can provide therapy for a wide varietyof tumors and cancers including skin cancers, connective tissue cancers,adipose cancers, breast cancers, lung cancers, stomach cancers,pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers,anogenital cancers, kidney cancers, bladder cancers, colon cancers,prostate cancers, central nervous system (CNS) cancers, retinal cancer,blood, and lymphoid cancers.

An “effective amount” as defined herein in relation to the treatment ofcancers is an amount that will decrease, reduce, inhibit, or otherwiseabrogate the growth of a cancer cell or tumor. In some embodiments, thecompound of Formula I, Formula II, or Formula III can be deliveredregionally to a particular affected region or regions of the subject'sbody. In some embodiments, wherein such treatment is considered moresuitable, the compound can be administered systemically. For example,the compound can be administered orally or intravenously.

In addition, it will be appreciated that therapeutic benefits for thetreatment of cancer can be realized by combining treatment with acompound or compounds of the compounds of the present invention with oneor more additional anti-cancer agents or treatments. The choice of suchcombinations will depend on various factors including, but not limitedto, the type of disease, the age and general health of the subject, theaggressiveness of disease progression, and the ability of the subject totolerate the agents that comprise the combination.

Thus, a variety of chemical compounds, also described as“anti-neoplastic” agents or “chemotherapeutic agents” can be used incombination with one or more of the novel anti-cancer compounds of thepresently described subject matter. Such compounds include, but are notlimited to, alkylating agents, DNA intercalators, protein synthesisinhibitors, inhibitors of DNA or RNA synthesis, DNA base analogs,topoisomerase inhibitors, anti-angiogenesis agents, and telomeraseinhibitors or telomeric DNA binding compounds. For example, suitablealkylating agents include alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as a benzodizepa,carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines,such as altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim. Inhibitorsof DNA synthesis, including alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards, intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining, and agents, such asdistamycin and netropsin, also can be combined with compounds of thecompounds of the present invention in pharmaceutical compositions.Topoisomerase inhibitors, such as coumermycin, nalidixic acid,novobiocin, and oxolinic acid, inhibitors of cell division, includingcolcemide, colchicine, vinblastine, and vincristine; and RNA synthesisinhibitors including actinomycin D, α-amanitine and other fungalamatoxins, cordycepin (3′-deoxyadenosine), dichlororibofuranosylbenzimidazole, rifampicine, streptovaricin, and streptolydigin also canbe combined with the inhibitor compounds of the compounds of the presentinvention to provide a suitable cancer treatment.

Thus, current chemotherapeutic agents that can be used in a combinationtreatment with the PFKFB3 inhibitor compounds of the present inventioninclude, but are not limited to, adrimycin, 5-fluorouracil (5FU),etoposide, camptothecin, actinomycin-D, mitomycin, cisplatin, hydrogenperoxide, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chjlorambucil, bisulfan, nitrosurea,dactinomycin, duanorubicin, doxorubicin, bleomycin, plicomycin,tamoxifen, taxol, transplatimun, vinblastin, and methotrexate, and thelike.

Combination treatments involving compounds of the present invention canbe tested and another therapeutic agent, such as anotherchemotherapeutic agent can be achieved by using both agents at the sametime. Alternatively, treatment with compounds of the present inventioncan precede or follow treatment with the other agent by intervalsranging from minutes to weeks.

The compounds of the present invention can be tested to measure theirability to inhibit growth of cancer cells, to induce apoptosis of thecancer cells, to reduce tumor burden, and to inhibit metastases. Forexample, one can measure cell growth according to the MTT assay, theAlamar Blue assay or the cell Titer Glow assay. Growth assays asmeasured by the methods listed above are well known in the art andmeasure either directly the number of viable cells (MTT and Alamar Blueassays) or the intracellular levels of ATP, inferring from the viabilityof cells (Cell Titer glow assay).

Accordingly, in one embodiment, a compound or its tautomeric,enantiomeric or diastereomeric form or a pharmaceutically acceptablesalt, prodrug, or metabolite thereof is provided, said compound havingthe formula:

wherein:

A, B, D, E, G and J are independently selected from the group consistingof N or C substituted with one of R₂, R₃, R₄, R₅, R₆ or R₇, wherein ifA, B, D, E, G, or J are N, then R₂, R₃, R₄, R₅, R₆ or R₇ represent afree electron pair at the N atom.

In compounds of Formula I, R₁ is selected from the group consisting ofhydrogen and linear or branched (C₁-C₆)-alkyl. In a more specificembodiment, R₁ is hydrogen or linear or branched (C₁-C₃)-alkyl. In amore specific embodiment, R₁ is hydrogen or methyl.

In compounds of Formula I, R₂, R₃, R₄, R₅, R₆ and R₇ are independentlyof one another, when attached to N, a free electron pair; or, whenattached to C, are selected from the group consisting of hydrogen,halogen, —COOH, linear or branched (C₁-C₆)-alkyl, linear or branched(C₁-C₆)-alkoxy, hydroxyl, —NH₂, N—(C₁-C₆)-alkyl, N-di-(C₁-C₆)-alkyl, and—SO₂CH₃. In a more specific embodiment, when attached to C, R₂, R₃, R₄,R₅, R₆ and R₇ are selected from the group consisting of hydrogen,halogen, —COOH, linear or branched (C₁-C₃)-alkyl, linear or branched(C₁-C₃)-alkoxy, hydroxyl, —NH₂, N—(C₁-C₃)-alkyl, N-di-(C₁-C₃)-alkyl, and—SO₂CH₃.

In compounds of Formula I, R₈ is selected from the group consisting ofhydrogen and linear or branched (C₁-C₆)-alkyl. In a more specificembodiment, R₈ is selected from the group consisting of hydrogen andlinear or branched (C₁-C₃)-alkyl.

In compounds of Formula I, X is

In compounds of Formula I, Y is selected from the group consisting ofsubstituted or unsubstituted (C₆-C₁₄)-aryl, substituted or unsubstituted(C₁-C₁₃)-heteroaryl having at least one of N, N═O, NH, N—(C₁-C₆)-alkyl,O, or S as ring members, and substituted or unsubstituted(C₃-C₈)-cycloalkyl. In a specific embodiment, the ring member is N orNH. In another specific embodiment, when Y is substituted (C₆-C₁₄)-aryl,substituted (C₁-C₁₃)-heteroaryl, or substituted (C₃-C₈)-cycloalkyl,substituents are selected from the group consisting of halogen, linearor branched (C₁-C₆)-alkyl, linear or branched (C₁-C₆)-alkoxy, hydroxyl,—OBn, —COOMe, —COOH, —NH₂, —CH₂OH, N—(C₁-C₆)-alkyl, N-di-(C₁-C₆)-alkyl,and —N—SO₂CH₃. In another specific embodiment, when Y is substituted(C₆-C₁₄)-aryl, substituted (C₁-C₁₃)-heteroaryl, or substituted(C₃-C₈)-cycloalkyl, substituents are selected from the group consistingof fluorine, chlorine, bromine, iodine, methyl, and methoxy.

In another embodiment, a pharmaceutical composition for the treatment ofcancer is provided, the composition comprising a compound according toFormula I and at least one pharmaceutically acceptable carrier. Inanother embodiment, the pharmaceutical composition further comprises oneor more additional chemotherapeutic agents.

In another embodiment, a method of treating cancer is provided, themethod comprising administering to a subject in need thereof aneffective amount of a compound according to Formula I.

In another embodiment, a method of treating a tumor is provided, themethod comprising administering to a subject in need thereof aneffective amount of a compound according to Formula I.

In still another embodiment, a method of inhibiting glycolytic flux in acell is provided, the method comprising contacting the cell with aneffective amount of a compound according to Formula I.

In another embodiment, a method of inhibiting enzymatic activity ofPFKFB3 in a cell is provided, the method comprising contacting the cellwith an effective amount of a compound according to Formula I.

EXAMPLES

The following examples are given by way of illustration only and are inno way intended to limit the scope of the present invention.

Example 1 Inhibition of Cancer Cell Proliferation

The ability of the compounds described herein to kill or inhibit theproliferation of cancer cells was measured using either the MTT assay,or the Alamar Blue assay, or the Cell Titer Glow® assay using 48 or 72hours exposure. Results for different cancer cell lines are shown in theTables 2, 3, and 4 below and demonstrate that these compounds inhibitcancer cell proliferation at low nanomolar concentrations across manytypes of cancer cell lines. The procedures are briefly described below.Cells of the desired tumor cell line were plated at 2×10⁵ cells/ml in 96well plates. Twice the indicated concentrations of the compounds of theinvention were added to cells the following day in an equal volume ofmedia. 72 hours later, cells were lysed and subjected to ATPdetermination using the CellTiter Glo-Luminescent Cell Viability Assaykit (Promega, Madison, Wis.). Experiments were done in triplicate. Whenusing the MTT assay or the Alamar blue assay, the experimentalconditions are essentially similar; at the end of the incubation period,20 microliters of the MTT solution is added per well and the samples areincubated for an additional four hours, rinsed, and the absorbance at570 nm measured. Results for the inhibition of cells proliferation arereported as IC₅₀ values as commonly done (the concentration leading to a50% inhibition of proliferation of the cell population) and are listedin the Tables below. Table 2 lists the IC₅₀ values for the compounds ofthe invention in several cell lines including Jurkat, Calu-6, NCI-H82,U937, HCT-116, and MDA-MB-231 cancer cell lines. Table 3 lists the IC₅₀of PFK-095 in a panel of 13 cell lines and Table 4 lists values forPFK-112 in a different panel of 10 cell lines.

TABLE 2 IC₅₀ values (Alamar Blue assay, 72 hours) NCl- HCT- MDA- JurkatSTD Calu-6 STD H82 STD U937 STD 116 STD MB-231 STD ACT- 0.775 0.3860.267 0.211 0.283 0.057 0.165 0.298 1.313 0.792 0.625 0.247 PFK- 065ACT- 1.500 0.566 0.208 0.035 0.498 23.087  0.050 0.007 2.400 0.141 0.3950.120 PFK- 095 ACT- 0.440 0.014 0.390 0.064 0.305 0.035 0.020 0.0000.850 0.212 — PFK- 095HCl ACT- 1.500 0.141 0.020 0.014 0.155 0.049 0.0140.003 0.695 0.120 — PFK- 096 ACT- 0.405 0.106 0.220 0.014 0.210 0.0000.040 0.000 0.490 0.127 — PFK- 098 ACT- 0.410 0.014 0.230 0.014 0.6800.014 0.076 0.008 0.495 0.064 — PFK- 099 ACT- >100 — >100 — >100 — >100— — — PFK- 101 ACT- >100 — >100 — >100 — >100 — — — PFK- 102 ACT- 0.1100.071 0.130 0.028 0.405 0.049 — 0.360 0.071 6.400 1.838 PFK- 103 ACT-0.118 0.032 0.200 0.014 0.310 0.028 — 0.320 0.014 5.950 0.283 PFK- 104ACT- 0.115 0.049 0.140 0.000 0.395 0.007 0.070 0.000 0.355 0.021 7.4001.131 PFK- 105 ACT- 21.000 5.657 8.900 1.131 15.000 2.828 3.150 0.07138.500 0.707 28.000 14.142  PFK- 106 ACT- 6.900 0.424 7.250 0.354 10.1006.223 2.850 0.212 >100 — 11.650 0.354 PFK- 107 ACT- 0.545 0.087 0.3600.113 0.360 0.057 0.205 0.010 0.475 0.096 0.090 0.000 PFK- 108 ACT- >100— 64.500 0.707 >100 — >100 — >100 — >100 — PFK- 109 ACT- >100 — 59.5000.014 >100 — >100 — >100 — >100 — PFK- 110 ACT- >100 0.141 57.50017.678  40.200 0.283 57.250 6.010 63.500 10.607 >100 — PFK- 111 ACT-0.625 0.320 0.260 0.139 0.685 0.096 0.130 0.008 1.443 0.590 0.205 0.021PFK- 112 ACT- 28.000 1.414 28.500 16.263  >100 — 26.800 1.697 >100— >100 0.120 PFK- 113 ACT- >100 — >100 — >100 — >100 — >100 — >100 —PFK- 114 ACT- 4.550 1.644 2.175 0.727 6.050 1.926 1.073 0.098 8.2003.732 0.620 0.240 PFK- 115 ACT- 0.125 0.021 0.080 0.028 0.205 0.0210.030 0.014 0.235 0.007 0.563 0.120 PFK- 116 ACT- 0.240 0.057 0.4200.028 0.385 0.021 0.095 0.007 1.700 0.424 0.673 0.163 PFK- 118 ACT-0.110 0.014 0.070 0.014 0.315 0.007 0.070 0.028 0.365 0.007 0.620 0.240PFK- 119 ACT- 0.155 0.007 0.055 0.007 0.270 0.021 — — — PFK- 120 ACT-0.115 0.021 0.079 0.013 0.335 0.014 — — — PFK- 121 ACT- 0.235 0.0140.070 0.014 0.320 0.028 — — — PFK- 122 ACT- 0.100 0.014 0.079 0.0130.315 0.007 — — — PFK- 123 ACT- 8.800 1.131 10.350 3.748 11.150 3.394 —— — PFK- 124 ACT- 22.700 0.990 39.500 1.131 20.300 1.131 — — — PFK- 125ACT- 0.850 0.071 1.100 0.141 0.840 0.057 — — — PFK- 126 ACT- 0.780 0.1061.020 0.042 0.755 0.078 — — — PFK- 127 ACT- >100 — >100 — >100 — — — —PFK- 128 ACT- 0.230 0.057 0.355 0.028 0.370 0.007 — — — PFK- 129 ACT-1.130 0.262 1.650 0.071 1.450 0.354 — — — PFK- 130 ACT- >100 — >100— >100 — — — — PFK- 131 ACT- 10.100 0.141 10.650 0.212 9.650 0.071 — — —PFK- 132 ACT- 2.350 0.778 40.850 0.212 11.900 1.414 — — — PFK- 133 ACT-3.300 1.131 2.450 0.212 3.450 0.636 — — — — PFK- 134

TABLE 3 IC₅₀ values (MTT assay, 72 hours) in a panel of cell lines usingPFK-095 PFK-095 Cell line IC₅₀ (microM) K562 0.21 MDA-MB-231 24.9 Jurkat4.65 H209 0.19 Hela 9.1 HT-29 0.45 HCT-116 0.51 U937 0.05 NCI-H82 0.18Calu-6 0.14 Hep G2 5.5 DU145 0.94 PC3 0.95

TABLE 4 IC₅₀ values (MTT assay, 48 hours) in a panel of cell lines usingPFK-112 PFK-112 Cell line IC₅₀ (nM) H22 513 CT26 1954 U87MG 3786 SK-N-SH305 LnCap 1030 A549 822 MiaPaca 154 BX-PC-3 135 BT474 1370 SK-BR-3 1112

Example 2 Inhibition of PFKFB3, the Recombinant Protein

The inducible bifunctional 6-Phosphofructo-2-kinase/fructose2,6-biphosphatase 3 enzyme (PFKFB3) was expressed and purified in orderto determine if the compounds of the invention inhibit its enzymaticactivity. PFKFB3 was prepared by expression in E. coli and purified byGST column and column chromatography. SDS Page coumassie staining gelsindicated that purity was high (>95%). The recombinant protein was pureand active as determined by the results of a kinase activity assaycommercially available (such as ADP Glow® assay, InVitrogen). The sameassay was used to determine the inhibition of the protein and theresults are shown in Table 5 below (IC₅₀ values or percent inhibition ata fixed concentration). Results confirmed that the compounds of theinvention have low nanomolar IC₅₀ values, interacting with PFKFB3 andinhibiting its enzymatic activity.

TABLE 5 PFKFB3 Inhibition results at a fixed compound concentration (250nM). % Inhibition of PFKFB3 Enzymatic Activity, 250 nM analogconcentration PFK065 PFK095 PFK096 PFK100 PFK111 PFK112 PFK116 PFK118PFK119 PFK123 % 13.5 26.2 0 0 54.3 81.5 34.2 0 3.7 19.4 Inhib.

Example 3 Microsomal Stability in Human Liver Microsomes

Microsomal stability in vitro in different biologic media (livermicrosomes, S9 fraction, hepatocytes) and species is ascertained toevaluate the rate at which a compound undergoes metabolism in theexperimental in vitro conditions. Results for some compounds of theinvention after a 60 minutes incubation period in human liver microsomesare shown in the Table 6 below. Results show that the rate of metabolismin vitro can be controlled as substitutions lead to metabolism ratesvarying between 20% and 100%. Experimental conditions are brieflydescribed here: the stability in human liver microsomes of severalcompounds was done over 24 hours at 37° C. using pooled mixed genderhuman liver microsomes. These liver microsomes were prepared at 1.0mg/ml of microsomal protein in a 100 mM potassium phosphate pH 7.4buffer with 1 mM NADPH. The media was incubated at 37° C. with thecompound in solution in DMSO. The concentration of the compound wasfollowed by LC/MS-MS as a function of time. Samples were assayed at t=0,15, 30, and 60 minutes. Testosterone was used as a positive control. Thesame experiment was performed with mouse, rat or dog liver microsomesinstead of human liver microsomes.

TABLE 6 Human Liver Microsomal stability results. HLM, Mean metabolized(%) Compound 0 min 60 min ACT-PFK-065 0.0 93 ACT-PFK-093 0.0 95ACT-PFK-095 0.0 26 ACT-PFK-096 0.0 99 ACT-PFK-104 0.0 80 ACT-PFK-105 0.051.4 ACT-PFK-107 0.0 100.0 ACT-PFK-108 0.0 73.3 ACT-PFK-112 0.0 82.7ACT-PFK-115 0.0 99.6 ACT-PFK-116 0.0 45.8 ACT-PFK-118 0.0 22.2ACT-PFK-119 0.0 79.2

Example 4 Cell Permeability

The determination of the permeability in Caco2 or MDR1-MDCK cells iswidely used when screening compounds to assess cell permeability,intestinal drug transport and predict absorption rates and to ascertainif a compound might be a Pgp substrate and have cross the blood brainbarrier. High permeability is determined as P_(app)>10×10⁻⁶ cm/s andsuggests that a compound might be orally absorbed. Comparison of theapical to basal (A-B) and basal to apical (B-A) values show if acompound is actively effluxed out the cells and if it is a Pgpsubstrate. Results for several compounds are shown below and suggestthat the compounds of the invention have high permeability properties,are not Pgp substrates, may be orally bioavailable and may cross theblood brain barrier. Briefly, cell permeability and transport mechanismsin Caco-2 and MDR1-MDCK monolayers experiments were performed intriplicate in the apical-to-basolateral and basolateral-to-apicaldirection using Transwell wells containing either Caco-2 or MDR1-MDCKmonolayers. A modified Hanks buffer pH 7.4 was used in both reservoirand receiver wells with the addition of 1% BSA in the receiver side.Confluent monolayers were used and their integrity was verified usingreference compounds (Atenolol as a low permeability reference compoundand Propanolol as a high permeability reference compound). A sample inthe basolateral and apical sides was taken after 2 hours and theconcentration measured by LC/MS-MS. Results are summarized in Tables 7Aand 7B below. The results also suggested that these compounds are notP-gp substrates and that they may cross the blood brain barrier.

TABLE 7A Permeability values in Caco2 cells P_(app) (cm/sec 10⁻⁶)Compounds (A-B) (B-A) ACT-PFK-065 10.3 5.9 ACT-PFK-095 16.2 8.9

TABLE 7B Permeability values in MDR1-MDCK cells ACT-PFK-112 7.8 17.3(cm/sec 10

) (A-B) (B-A)

indicates data missing or illegible when filed

Example 5 Solubility Measurements

Chemical properties of the compounds of the invention were investigated,including the solubility in several solvents and pharmaceuticallyaccepted excipients. Results show the solubility profile can bemodified, resulting in solubility values in the mg/ml range that aresatisfactory in order to treat cancer patients. Briefly, selectedcompounds of the invention are dissolved in a small volume of thesolvents/excipients of interest at room temperature over 1 hour intypically 1 ml and the concentration is measured by HPLC afterfiltration.

TABLE 8 Solubility results after 1 hour at room temperature Cpd EtOHAcetone DCM Cremophor Tween 80 Cre:EtOH¹ Twee80:EtOH² Cre:EtOH:Saline³PFK-095 1.6 5.0 3.4 6.9 7.4 10 9.7 0.3 PFK-096 2.8 25.8 8.9 1.2 1.8 29.718.7 1.7 PFK-098 4.6 9.3 10.4 30.2 37 29 32.8 2.4 PFK-108 9.9 12.8 11.137.6 16.9 78.1 10.3 4.8 PFK-112 7.4 8.2 14 18.6 18.1 37.6 35.6 3.7PFK-116 1.3 6.2 6 14.4 15.5 8.6 10.2 0.7 PFK-118 0.2 0.6 0.3 2.2 1.6 11.3 0.1 PFK-119 1 5.5 0.7 14.4 11.4 6.1 8.6 0.3 ¹Cre:EtOH is a 1:1cremophor EL-P:ethanol mixture ²Tween80:EtOH is a 6:4 mixture³Cre:EtOH:Saline is a 5:5:90 mixture

Example 6 Inhibition of 2-Deoxyglucose Uptake

Inhibition of PFKFB3 results in inhibiting glycolysis. Several feedbackor feedforward activation and inhibition mechanisms exist so that, by afeedback mechanism, inhibiting the activity of PFKFB3 may inhibitglucose uptake by cells. An assay was developed to determine if therewas inhibition of glucose uptake following exposure to the compounds onthe invention. Briefly, Jurkat cells were plated at 1×10⁵/mL in RPM′1640 supplemented with 10% fetal bovine serum and 50 μg/mL gentamicinsulfate. Cells were immediately treated with vehicle or 5 μmol/L of thecompound of the invention for 3 hours and subsequently placed inglucose-free RPMI 1640 for 30 min. ¹⁴C-2-deoxyglucose (0.25 μCi/mL;Perkin Elmer) was added for an additional 60 min and cells were thenwashed three with ice-cold RPMI 1640 containing no glucose. Cell lysateswere collected in 500 μL of 0.1% SDS, and scintillation counts(counts/min) were measured on 400 μL of lysate. Counts were normalizedto protein concentration.

Results shown in Table 9 indicate a rapid and quantitative inhibition ofglucose uptake by cells treated with PFKFB3 inhibitors.

TABLE 9 Percentage of Deoxyglucose Uptake Inhibition. % Inhibition in 2-Deoxyglucose Compound Concentration Uptake PFK-095 5 μM 48 PFK-096 5 μM29 PFK-098 5 μM 25 PFK-108 5 μM 2 PFK-109 5 μM 42 PFK-112 5 μM 47PFK-116 5 μM 0

Example 7 Pharmacokinetics

The pharmacokinetic parameters for several compounds were determined inmice following IV (intravenous), IP (intraperitoneal), or PO (per os)administration, as well as in rats and dogs. A typical study designincludes six Balbc male mice 7 to 8 weeks old. For instance, a dose of10 mg/kg was administered IV using a 5% DMS0/90% Captisol (20% in water)solution or other pharmaceutically acceptable parenteral and oralformulations. Blood samples were collected at different intervals.Plasma samples were extracted and analyzed using an LC-MS/MS method.Similar protocols were used for the rats (Sprague Dawley rats) or dogs(beagles) PK studies; in the case of dogs PK studies, the number ofanimals per group was three.

Results illustrated in FIGS. 1 to 3 show that the compounds of theinvention have good pharmacokinetic properties and that they are orallybioavailable with F (the oral bioavailability) varying between 25% forPFK-065 and 95% for PFK-112.

Table 10 shows the IV pharmacokinetic parameters for compounds dosedintravenously. PFK-065 was dosed at 10 mg/kg in male Balbc mice; PFK-095was dosed at 10 mg/kg in male Sprague Dawley rats; and PFK-112 dosed at6 mg/kg in male beagle dogs.

TABLE 10 IV PK parameters for PFK-065, PFK-095, and PFK-112 T_(1/2b)C_(max) AUC _(0-inf) Vd % F (h) (ng/mL) (ng · h/ml) (L/kg) (from POgroup) PFK-065 2.1 4112 2627 11.2 25% PFK-095 5.5 16932 1293 2.5 38%PFK-112 5.8 5065 5066 1.8 95%

Example 9 Efficacy Studies

The activity of several compounds of the invention was investigated invivo in tumor models. Several tumor models were used in these studies(the Lewis Lung carcinoma model, the human glioblastoma xenograft U87MGtumor model, the human colorectal carcinoma cancer model HCT-116, theCT-26 colon carcinoma cancer model, the Lung Calu-6 model, the BT474breast carcinoma model). The experimental protocol for the U87MG studyis described below. Subsequent to subcutaneous inoculation of tumorcells, tumors started to develop and once tumors reached the desiredvolume of 150 mm³ on average, treatment was initiated. Tumor volume wasmonitored in both groups and the average for both the control andtreatment groups three times a week, as well as body weights.

Briefly, athymic nude mice at 7-8 weeks of age were used for the study.Mice were housed in microisolator housing, with food and water providedas libitum, and quarantined for 4 days prior to the initiation of thestudy. U87MG cells were maintained in McCoy's 5A medium supplementedwith 10% fetal bovine serum and 2 mM glutamine. Cells at 80% confluencewere harvested using 0.25% trypsin/EDTA solution, washed once with PBSand resuspended in a mixture of serum-free medium/Matrigel (1:1 byvolume) at a density of 3×10 6 cells/100 μl. 4 groups of 10 mice eachwere used in that experiment. U87MG cells suspended in 100 μl of amixture of medium/Matrigel (1:1) were subcutaneously implanted in theright flank region. Animals were monitored for tumor growth daily aftercell implantation. When tumor volumes reached approximately 150 mm3,mice were randomized into 2 groups of 8 mice each using only mice havingtumor volumes closest to the mean value. Tumor volumes were measuredusing the formula V=L×W×H×π/6, where L and W represent the longer andshorter diameters of the tumor and H represents the height of the tumor.Treatment began following randomization. ACT-PFK-112 was administered IVat a dose of 50 mg/kg on days 1, 2, 3, 7, 8, 9, 13, 14, and 15. Animalswere observed for possible toxic effect from the drug treatment. Bodyweights were recorded and showed that the compounds were very welltolerated.

Similar protocols were used for the other studies using differentmodels;

differences include the use of normal mice (C57B1/6); administeringlarger number of cancer cells; including a positive control group;dosing using different routes of administration and different schedulesor doses.

Results of several studies are shown in FIGS. 4 to 6 and demonstratethat compounds of the invention effectively inhibit tumor growth in vivoin different tumor types.

All documents cited are incorporated herein by reference; the citationof any document is not to be construed as an admission that it is priorart with respect to the present invention.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to one skilled in the artthat various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A compound or its tautomeric, enantiomeric or diastereomeric form ora pharmaceutically acceptable salt, prodrug, or metabolite thereof, saidcompound having the formula:

wherein: A, B, D, E, G and J are independently selected from the groupconsisting of N or C substituted with one of R₂, R₃, R₄, R₅, R₆ or R₇,wherein if A, B, D, E, G, or J are N, then R₂, R₃, R₄, R₅, R₆ or R₇represent a free electron pair at the N atom; R₁ is selected from thegroup consisting of hydrogen and linear or branched (C₁-C₆)-alkyl; R₂,R₃, R₄, R₅, R₆ and R₇ are independently of one another, when attached toN, a free electron pair, or, when attached to C, are selected from thegroup consisting of hydrogen, halogen, —COOH, linear or branched(C₁-C₆)-alkyl, linear or branched (C₁-C₆)-alkoxy, hydroxyl, —NH₂,N—(C₁-C₆)-alkyl, N-di-(C₁-C₆)-alkyl, and —SO₂CH₃; R₈ is selected fromthe group consisting of hydrogen and linear or branched (C₁-C₆)-alkyl;

and Y is selected from the group consisting of substituted orunsubstituted (C₆-C₁₄)-aryl, substituted or unsubstituted(C₁-C₁₃)-heteroaryl having at least one of N, N═O, NH, N—(C₁-C₆)-alkyl,O, or S as ring members, and substituted or unsubstituted(C₃-C₈)-cycloalkyl.
 2. The compound of claim 1, wherein when Y issubstituted (C₆-C₁₄)-aryl, substituted (C₁-C₁₃)-heteroaryl, orsubstituted (C₃-C₈)-cycloalkyl, substituents are selected from the groupconsisting of halogen, linear or branched (C₁-C₆)-alkyl, linear orbranched (C₁-C₆)-alkoxy, hydroxyl, —OBn, —COOMe, —COON, —NH₂, —CH₂OH,N—(C₁-C₆)-alkyl, N-di-(C₁-C₆)-alkyl, and —N—SO₂CH₃.
 3. The compound ofclaim 2, wherein when Y is substituted (C₆-C₁₄)-aryl, substituted(C₁-C₁₃)-heteroaryl, or substituted (C₃-C₈)-cycloalkyl, substituents areselected from the group consisting of fluorine, chlorine, bromine,iodine, methyl, and methoxy.
 4. The compound of claim 1, wherein R₁ ishydrogen or methyl.
 5. A pharmaceutical composition for the treatment ofcancer comprising a compound according to claim 1 and at least onepharmaceutically acceptable carrier.
 6. The pharmaceutical compositionof claim 5, further comprising one or more additional chemotherapeuticagents.
 7. A method of treating cancer comprising administering to asubject in need thereof an effective amount of a compound according toclaim
 1. 8. A method of treating a tumor, comprising administering to asubject in need thereof an effective amount of a compound according toclaim
 1. 9. A method of inhibiting glycolytic flux in a cell, comprisingcontacting the cell with an effective amount of a compound according toclaim
 1. 10. A method of inhibiting enzymatic activity of PFKFB3 in acell, comprising contacting the cell with an effective amount of acompound according to claim
 1. 11. A compound selected from the groupset forth in Table
 1. 12. A compound selected from the group consistingof: (3H-Benzo[e]indol-2-yl)-pyridin-4-yl-methanone;(3H-Benzo[e]indol-2-yl)-(2-methoxy-phenyl)-methanone;(3H-benzo[e]indol-2-yl)-(2-chloro-pyridin-4-yl)-methanone;(3H-Benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy-phenyl)-methanone;(4-Amino-phenyl)-(3H-benzo[e]indol-2-yl)-methanone;(3H-Benzo[e]indol-2-yl)-(2-methoxy-pyridin-4-yl)-methanone;(5-Fluoro-3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanone;(5-Fluoro-3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy-phenyl)-methanone;(3H-Benzo[e]indol-2-yl)-(3-hydroxymethyl-phenyl)-methanone;(5-Fluoro-3H-benzo[e]indol-2-yl)-(3-fluoro-4-hydroxy-phenyl)-methanone;(3H-Benzo[e]indol-2-yl)-p-tolyl-methanone;(4-Amino-3-methoxy-phenyl)-(3H-benzo[e]indo)-2-yl)-methanone;(4-Amino-3-methoxy-phenyl)-(5-hydroxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-3-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone;N-[4-(3H-Benzo[e]indole-2-carbonyl)-phenyl]-methanesulfonamide;(4-Amino-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-2-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-3-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-2-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-3-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-2-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-3-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-2-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-3-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone;(4-Amino-2-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone;(4-Amino-phenyl)-(7-methoxy-3H-benzo[e]indol-2-yl)-methanone;(4-Amino-phenyl)-(5-hydroxy-3-methyl-3H-benzo[e]indol-2-yl)-methanone;(7-Amino-5-fluoro-9-hydroxy-3H-benzo[e]indol-2-yl)-(3-methyl-pyridin-4-yl)-methanone;(5-Amino-3H-pyrrolo[3,2-f]isoquinolin-2-yl)-(3-methoxy-pyridin-4-yl)-methanone;(4-Amino-2-methyl-phenyl)-(9-hydroxy-3H-pyrrolo[2,3-e]quinolin-2-yl)-methanone;and(4-Amino-phenyl)-(7-methanesulfonyl-3H-benzo[e]indol-2-yl)-methanone.